The physiological role of opioid neurotransmission remains unclear. Part of this lack of clarity is due to uncertainties concerning the volume of extracellular space through which opioid peptides must diffuse to reach their receptors. For a variety of reasons it now seems that the classical concept of the synapse is not an appropriate model in which to explore the physiological role of opioids. Most importantly, it is very unlikely that the active zone of presynaptic nerve terminals (the site from which "classical" transmitters are released by exocytosis) can be the site from which opioids are released. The purpose of this proposal is to determine at the cellular and subcellular level the sites at which opioid peptides are released from axons. Classical, small molecule neurotransmitters are stored in small synaptic vesicles; the opioids and other neuropeptides are preferentially stored in large granular vesicles. Independent mechanisms exist which are responsible for intracellular trafficking and exocytosis of these two populations of vesicles. In this proposal, experiments are proposed which will determine the spatial occurrence of molecules directly involved in the release of large granular vesicles. The following studies will be conducted: 1. The spatial occurrence of N- and L-types of calcium channels within the membranes of opioidergic nerve fibers in model neuronal systems will be determined using fluorescently-labeled omegaconotoxin, nisoldipine and funnel web spider toxin omega-Aga-IIIA. 2. The spatial occurrence of the alpha1 subunit of the rat brain L-type of calcium channel within the membranes of opioidergic nerve fibers in the mouse vas deferens and the enteric nervous system of the guinea pig ileum will be determined using antibodies selective for peptide sequences of this subunit. 3. The spatial occurrence of calpactin, the putative docking protein for large granular vesicles, will be localized with respect to opioidergic nerve fibers and terminals in the model neural systems. 4. Certain small GTP-binding proteins will be localized with respect to opioidergic nerve fibers, since it is likely that at least one member of this family is crucial in the final stages of large granular vesicle fusion and release.